Detection of Bordetella

ABSTRACT

The invention provides methods to detect  Bordetella pertussis  and/or  Bordetella parapertussis  in a biological sample. Primers and probes for the differential detection of  B. pertussis  and  B. parapertussis  are provided by the invention. Articles of manufacture containing such primers and probes for detecting  B. pertussis  and/or  B. parapertussis  are further provided by the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application and claims priority under35 U.S.C. §120 of U.S. application Ser. No. 10/754,223 filed Jan. 9,2004, which is a Continuation-in-Part application and claims priorityunder 35 U.S.C. §120 of U.S. application Ser. No. 10/062,875 filed Jan.31, 2002, which claims priority under 35 U.S.C. §119(e) of U.S.Application No. 60/265,534 filed Jan. 31, 2001.

TECHNICAL FIELD

This invention relates to bacterial diagnostics, and more particularlyto detection of Bordetella.

BACKGROUND

Whooping cough, caused by Bordetella pertussis, is presently one of theten most common causes of death from infectious disease worldwide.Patients first present with a common cold and a cough. However, thedisease progresses to paroxysmal coughing followed by a characteristicinspiratory whoop. Secondary symptoms arising from bacterial pneumonia,neurological complications (i.e., seizures and encephalopathy), andpressure effect complications (i.e., pneumothorax, epistaxis, subduralhematomas, hernias, and rectal prolapse) can also occur. From the onsetof initial symptoms, the disease can take 6-8 weeks to resolve.Bordetella parapertussis is closely related to B. pertussis and maycause a similar illness, especially in children; however, the symptomsare less severe and are generally of shorter duration than B. pertussis.

Pertussis and its associated complications were a major cause of infantand childhood mortality until the introduction of thediphtheria-tetanus-pertussis (DTP) vaccine in the 1940s. Widespread useof the vaccine in the American population resulted in a 98% decrease inthe incidence of pertussis. According to the Centers for Disease Control(CDC; Atlanta, Ga.), there has been a resurgence of pertussis, and theincidence of pertussis in the general population has been on the risesince 1991. There was an 82% increase in total cases reported to the CDCin 1993 compared to the same period in 1992 (1992=3004; 1993=5457). In1992, there were outbreaks of pertussis in Massachusetts and Maryland,and in 1994 there was an outbreak of erythromycin-resistant B. pertussisdescribed in Arizona. This trend is also being seen outside the UnitedStates. In 1996, the Netherlands had an outbreak of pertussis, reporting12 times the number of cases seen in 1995 (1995=341; 1996=4231).

Many cases of B. pertussis go undiagnosed and unreported. Whilepertussis is highly communicable and can cause severe disease, symptomsin older children and adults, including those previously immunized, maybe difficult to differentiate from the nonspecific symptoms ofbronchitis and upper respiratory tract infections. Clinical diagnosis ofpertussis is complicated by the fact that the characteristic cough(whoop) is rarely observed in infants and adult patients.

SUMMARY

Methods of the invention can be used to rapidly identify B. pertussisand/or B. parapertussis from a biological sample for differentialdiagnosis of pertussis infection. Nasopharyngeal swabs and aspirates canbe treated to release the DNA from Bordetella species in the sample.Using specific primers and probes, the method includes amplifying andmonitoring the development of specific template nucleic acid usingfluorescence resonance emission technology (FRET).

In one aspect, the invention provides a method for detecting thepresence or absence of Bordetella pertussis and/or B. parapertussis in abiological sample from an individual. The method includes performing atleast one cycling step of amplifying and hybridizing. The amplifyingstep includes contacting the sample with a pair of IS481 primers and/ora pair of IS1001 primers to produce an IS481 and/or an IS1001amplification product, respectively, if IS481 and/or IS1001 nucleic acidmolecules are present in the sample. The hybridizing step includescontacting the sample with a pair of IS481 probes and/or a pair ofIS1001 probes. Generally, the members within each pair of IS481 andIS1001 probes hybridize within no more than five nucleotides of eachother. Typically, a first IS481 probe of the pair of IS481 probes islabeled with a donor fluorescent moiety and a second IS481 probe of thepair of IS481 probes is labeled with a corresponding acceptorfluorescent moiety. Likewise, a first IS1001 probe of the pair of IS1001probes is labeled with a donor fluorescent moiety and a second IS1001probe of the pair of IS1001 probes is labeled with a correspondingacceptor fluorescent moiety. The donor fluorescent moiety and/or theacceptor fluorescent moieties on the IS481 and the IS1001 probes can bedifferent.

The method further includes detecting the presence or absence of FRETbetween the donor fluorescent moiety of the first IS481 probe and thecorresponding acceptor fluorescent moiety of the second IS481 probeand/or between the donor fluorescent moiety of the first IS1001 probeand the corresponding acceptor fluorescent moiety of the second IS1001probe. The presence of FRET usually indicates the presence of B.pertussis and/or B. parapertussis in the biological sample, while theabsence of FRET usually indicates the absence of B. pertussis or B.parapertussis in the biological sample.

The method can additionally include determining the melting temperaturebetween the IS481 probes and the IS481 amplification product and/orbetween the IS1001 probes and the IS1001 amplification product. Themelting temperature(s) further confirms the presence or absence of B.pertussis and the presence or absence of B. parapertussis in the sample.

In another aspect of the invention, the above-described method can beperformed to detect B. pertussis using primers and probes that hybridizeto IS481 nucleic acid molecules. Alternatively, the above-describedmethod can be performed to detect B. parapertussis using primers andprobes that hybridize to IS1001 nucleic acid molecules.

In one aspect of the invention, there is provided a pair of IS481primers including a first IS481 primer and a second IS481 primer. Afirst IS481 primer can include the sequence 5′-CCA GTT CCT CAA GGACGC-3′ (SEQ ID NO:1), and the second IS481 primer can include thesequence 5′-GAG TTC TGG TAG GTG TGA GCG TA-3′ (SEQ ID NO:2). A firstIS481 probe can include the sequence 5′-CAC CGC TTT ACC CGA CCT TAC CGCCCA C-3′ (SEQ ID NO:3), and a second IS481 probe can include thesequence 5′-GAC CAA TGG CAA GGC CGA ACG CTT CAT C-3′ (SEQ ID NO:4). Inanother embodiment, a second IS481 probe can include the sequence 5′-GACCAA TGG CAA GGC TCG AAC GCT TCA TC-3′ (SEQ ID NO:11).

In another aspect of the invention, there is provided a pair of IS1001primers including a first IS1001 primer and a second IS1001 primer. Afirst IS1001 primer can include the sequence 5′-GGC GAT ATC AAC GGGTGA-3′ (SEQ ID NO:5), and the second IS1001 primer can include thesequence 5′-CAG GGC AAA CTC GTC CAT C-3′ (SEQ ID NO:6). The inventionfurther provides a first IS1001 probe that can include the sequence5′-GTT CTT CGA ACT GGG TTG GCA TAC-3′ (SEQ ID NO:7), and a second IS1001probe that can include the sequence 5′-GTC AAG ACG CTG GAC AAG GCT C-3′(SEQ ID NO:8). In another embodiment, a first IS1001 probe can includethe sequence 5′-GGT TGG CAT ACC GTC AAG A-3′ (SEQ ID NO:12), and asecond IS1001 probe can include the sequence 5′-GCT GGA CAA GGC TCG-3′(SEQ ID NO:13).

Representative biological samples include nasopharyngeal swabs,nasopharyngeal aspirates, and throat swabs. Generally, the members ofthe pair of IS481 probes hybridize within no more than two nucleotidesof each other, or within no more than one nucleotide of each other. Arepresentative donor fluorescent moiety is fluorescein, andcorresponding acceptor fluorescent moieties include LCT™-Red 640,LCT™-Red 705, Cy5, and Cy5.5. Additional corresponding donor andacceptor fluorescent moieties are known in the art.

In one aspect, the detecting step includes exciting the biologicalsample at a wavelength absorbed by the donor fluorescent moiety andvisualizing and/or measuring the wavelength emitted by the acceptorfluorescent moiety. In another aspect, the detecting step includesquantitating FRET. In yet another aspect, the detecting step isperformed after each cycling step (e.g., in real-time).

The above-described methods can further include preventing amplificationof a contaminant nucleic acid. Preventing amplification can includeperforming amplifying steps in the presence of uracil and treating thebiological samples with uracil-DNA glycosylase prior to amplifying. Inaddition, the cycling step can be performed on a control sample. Acontrol sample can include the same portion of the IS481 or IS1001nucleic acid molecule. Alternatively, a control sample can include anucleic acid molecule other than an IS481 or IS1001 nucleic acid.Cycling steps can be performed on such a control sample using a pair ofcontrol primers and a pair of control probes that are other than IS481or IS1001 primers and probes. One or more amplifying steps produces acontrol amplification product. Each of the control probes hybridize tothe control amplification product.

In yet another aspect, the invention provides articles of manufacture,or kits. Kits of the invention can include a pair of IS481 primers, apair of IS481 probes, and a donor and corresponding acceptor fluorescentmoiety. For example, a first IS481 primer provided in a kit of theinvention can include the sequence 5′-CCA GTT CCT CAA GGA CGC-3′ (SEQ IDNO:1), and a second IS481 primer can include the sequence 5′-GAG TTC TGGTAG GTG TGA GCG TA-3′ (SEQ ID NO:2). A first IS481 probe provided in akit of the invention can include the sequence 5′-CAC CGC TTT ACC CGA CCTTAC CGC CCA C-3′ (SEQ ID NO:3), and a second IS481 probe can include thesequence 5′-GAC CAA TGG CAA GGC CGA ACG CTT CAT C-3′ (SEQ ID NO:4). Inanother embodiment, a second IS481 probe provided in a kit of theinvention can include the sequence 5′-GAC CAA TGG CAA GGC TCG AAC GCTTCA TC-3′ (SEQ ID NO:11).

In another aspect of the invention, there is provided an article ofmanufacture, or kit. Kits of the invention can include a pair of IS481primers, a pair of IS481 probes, and a donor and corresponding acceptorfluorescent moiety. For example, a first IS1001 primer provided in a kitof the invention can include the sequence 5′-GGC GAT ATC AAC GGG TGA-3′(SEQ ID NO:5), and a second IS1001 primer can include the sequence5′-CAG GGC AAA CTC GTC CAT C-3′ (SEQ ID NO:6). A first IS1001 probeprovided in a kit of the invention can include the sequence 5′-GTT CTTCGA ACT GGG TTG GCA TAC-3′ (SEQ ID NO:7), and the second IS1001 probecan include the sequence 5′-GTC AAG ACG CTG GAC AAG GCT C-3′ (SEQ IDNO:8). In another embodiment, a first IS1001 probe provided in a kit ofthe invention can include the sequence 5′-GGT TGG CAT ACC GTC AAG A-3′(SEQ ID NO:12), and a second IS1001 probe provided in a kit of theinvention can include the sequence 5′-GCT GGA CAA GGC TCG-3′ (SEQ IDNO:13).

Articles of manufacture can include fluorophoric moieties for labelingthe probes or probes already labeled with donor and correspondingacceptor fluorescent moieties. The article of manufacture can alsoinclude a package insert having instructions thereon for using theprimers, probes, and fluorophoric moieties to detect the presence orabsence of Bordetella in a biological sample and can further includeinstructions thereon for using the probes to distinguish between B.pertussis and/or B. parapertussis in a biological sample.

In yet another aspect of the invention, there is provided a method fordetecting the presence or absence of B. pertussis in a biological samplefrom an individual. Such a method includes performing at least onecycling step. A cycling step can include an amplifying step and ahybridizing step. Generally, an amplifying step includes contacting thesample with a pair of IS481 primers to produce an IS481 amplificationproduct if a B. pertussis IS481 nucleic acid molecule is present in thesample. Generally, a hybridizing step includes contacting the samplewith an IS481 probe. Such an IS481 probe is usually labeled with a donorfluorescent moiety and a corresponding acceptor fluorescent moiety. Themethods further include detecting the presence or absence offluorescence resonance energy transfer (FRET) between the donorfluorescent moiety and the acceptor fluorescent moiety of the IS481probe. The presence or absence of FRET is indicative of the presence orabsence of B. pertussis in said sample. In addition to the IS481 primersand probe described herein, this method also can be performed usingIS1001 primers and probe.

In one aspect, amplification can employ a polymerase enzyme having 5′ to3′ exonuclease activity. Thus, the donor and acceptor fluorescentmoieties would be within no more than 5 nucleotides of each other alongthe length of the probe. In another aspect, the IS481 probe includes anucleic acid sequence that permits secondary structure formation. Suchsecondary structure formation generally results in spatial proximitybetween the donor and corresponding acceptor fluorescent moiety.According to this method, the acceptor fluorescent moiety on a probe canbe a quencher.

In another aspect of the invention, there is provided a method fordetecting the presence or absence of B. pertussis in a biological samplefrom an individual. Such a method includes performing at least onecycling step. A cycling step can include an amplifying step and adye-binding step. An amplifying step generally includes contacting thesample with a pair of IS481 primers to produce an IS481 amplificationproduct if a B. pertussis IS481 nucleic acid molecule is present in thesample. A dye-binding step generally includes contacting the IS481amplification product with a nucleic acid binding dye. The methodfurther includes detecting the presence or absence of binding of thenucleic acid binding dye to the amplification product. According to theinvention, the presence of binding is typically indicative of thepresence of B. pertussis in the sample, and the absence of binding istypically indicative of the absence of B. pertussis in the sample. Sucha method can further include the steps of determining the meltingtemperature between the IS481 amplification product and the nucleic acidbinding dye. Generally, the melting temperature confirms the presence orabsence of B. pertussis. Representative double-stranded DNA binding dyesinclude SYBRGreenI®, SYBRGold®, and ethidium bromide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

DETAILED DESCRIPTION

B. pertussis, the bacterium causing pertussis or “whooping cough” hastraditionally been difficult to detect in a clinically useful manner.Several diagnostic methods are available, but most lack sensitivity,require extended culture incubation times for results, and/or requirerepeated sampling and testing to verify significant increases ofimmunoglobulin G antibodies against pertussis toxin or immunoglobulin Aantibodies against B. pertussis in paired sera. The present inventionprovides methods of detecting B. pertussis and/or B. parapertussis in abiological sample from an individual suspected of having pertussis. Themethods feature the ability to distinguish between B. pertussis and B.parapertussis. The invention further provides kits containing primersand probes to carry out the differential diagnostic methods of theinvention.

Pertussis

B. pertussis is transmitted by respiratory droplets and causes diseaseonly in humans. Virulence factors of B. pertussis include agglutinogens,fimbriae, P.69/pertactin, pertussis toxin, filamentous haemagglutinin,adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin,lipopolysaccharide, tracheal colonization factor, serum resistancefactor, and type III secretion. Virulence factor expression is regulatedby the bvgAS locus, a two-component signal transduction system. Thepathophysiological sequence consists of attachment (fimbriae,P.69/pertactin, tracheal colonization factor, pertussis toxin,filamentous haemagglutinin), evasion of host defense (adenylate cyclase,pertussis toxin, serum resistance factor), local effects (trachealcytotoxin), and systemic effects (pertussis toxin).

Various methods to diagnose pertussis are available, including culture,serological methods, and the polymerase chain reaction (PCR). Serotypingof isolates to detect agglutinogens 2 and 3 is useful because serotype1,2 may be associated with higher mortality, and antibodies to theagglutinins may be protective in both animals and humans. Acellularvaccines containing one to five components are increasingly being usedin various countries. Immunization using whole-cell vaccine is alsoeffective but is reactogenic. Protective immunity to pertussiscorrelates with high levels of antibody to each of pertactin, fimbriae,and pertussis toxin.

Pertussis is a communicable disease that can be very severe in younginfants. Early diagnosis and treatment are essential to limit theseverity of the disease and minimize transmission. The wide prevalenceof pertussis and its changing epidemiology has highlighted the need formore sensitive and rapid methods for diagnostic testing. Currentdiagnostic tests for B. pertussis and B. parapertussis are difficult toperform due to the fastidious nature of Bordetella organisms, lacksensitivity, and require 3-5 days of growth to allow identification.Serologic testing by enzyme-linked immunosorbent assay (ELISA) orWestern blot is sensitive and specific, but requires the comparison of 2serum specimens from the subject collected over a 4-week interval.Direct fluorescent antibody testing (DFA) of nasopharyngeal secretionslacks sensitivity. The reference method is direct culture of theorganism from nasopharyngeal secretions, but direct culture ofBordetella has a turnaround time of 1 to 2 days. Further, the organismis susceptible to environmental exposure (changes in temperature anddrying) and has specific growth requirements, making recovery by culturedifficult.

B. pertussis and B. parapertussis Nucleic Acids and Oligonucleotides

In one embodiment, methods of the invention use the insertion sequenceIS481 (GenBank Accession No. M28220; SEQ ID NO:9) to detect B. pertussisin a biological sample. B. pertussis typically contains 50-100 copies ofIS481. The IS481 sequence was described by McPheat et al. (J. Gen.Microbiol., 135:1515-1520, 1989). In another embodiment, methods of theinvention use the insertion sequence IS1001 (GenBank Accession No.X66858; SEQ ID NO:10) to detect B. parapertussis in a biological sample.B. parapertussis typically contains 30-35 copies of IS1001. The IS1001sequence was described by van der Zee et al. (J. Bacteriol.,175:141-147, 1993). Bordetella nucleic acids other than thoseexemplified herein (e.g., other than IS481 or IS1001 nucleic acids) alsocan be used to detect Bordetella in a sample and are known to those ofskill in the art. Specifically, primers and probes to amplify and detectB. pertussis IS481 nucleic acid are provided by the invention, as areprimers and probes to amplify and detect B. parapertussis IS1001 nucleicacid.

Primers that amplify a Bordetella nucleic acid molecule (e.g., IS481 orIS1001) can be designed using, for example, a computer program such asOLIGO (Molecular Biology Insights, Inc., Cascade, Colo.). Importantfeatures when designing oligonucleotides to be used as amplificationprimers include, but are not limited to, an appropriate sizeamplification product to facilitate detection (e.g., byelectrophoresis), similar melting temperatures for the members of a pairof primers, and the length of each primer (i.e., the primers need to belong enough to anneal with sequence-specificity and to initiatesynthesis but not so long that fidelity is reduced duringoligonucleotide synthesis). Typically, oligonucleotide primers are 8 to50 nucleotides in length (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).“IS481 primers” and “IS1001 primers” as used herein refers tooligonucleotide primers that specifically anneal to B. pertussis IS481nucleic acid sequences and B. parapertussis IS1001 nucleic acidsequences, respectively, and initiate synthesis therefrom underappropriate conditions.

Designing oligonucleotides to be used as hybridization probes can beperformed in a manner similar to the design of primers, although themembers of a pair of probes preferably anneal to an amplificationproduct within no more than 5 nucleotides of each other on the samestrand such that FRET can occur (e.g., within no more than 1, 2, 3, or 4nucleotides of each other). This minimal degree of separation typicallybrings the respective fluorescent moieties into sufficient proximitysuch that FRET can occur. It is to be understood, however, that otherseparation distances (e.g., 6 or more nucleotides) are possible providedthe fluorescent moieties are appropriately positioned relative to eachother (for example, with a linker arm) such that FRET can occur. Inaddition, probes can be designed to hybridize to targets that contain amutation or polymorphism, thereby allowing differential detection basedon either absolute hybridization of different pairs of probescorresponding to the particular species to be distinguished ordifferential melting temperatures between, for example, members of apair of probes and each amplification product corresponding to thespecies to be distinguished. As with oligonucleotide primers,oligonucleotide probes usually have similar melting temperatures, andthe length of each probe must be sufficient for sequence-specifichybridization to occur but not so long that fidelity is reduced duringsynthesis. Oligonucleotide probes are 8 to 50 nucleotides in length(e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, or 50 nucleotides in length). “IS481 probes” and “IS1001probes” as used herein refers to oligonucleotide probes thatspecifically anneal to a B. pertussis IS481 amplification product and aB. parapertussis IS1001 amplification product, respectively.

Constructs of the invention include vectors containing a Bordetellanucleic acid e.g., an IS481 or IS1001 nucleic acid molecule, or fragmentthereof. Constructs can be used, for example, as control templatenucleic acid molecules. Vectors suitable for use in the presentinvention are commercially available and/or produced by recombinant DNAtechnology methods routine in the art. IS481 or IS1001 nucleic acidmolecules can be obtained, for example, by chemical synthesis, directcloning from the respective Bordetella organism, or by PCRamplification. Constructs suitable for use in the methods of theinvention typically include, in addition to IS481 or IS1001 nucleic acidmolecules, sequences encoding a selectable marker (e.g., an antibioticresistance gene) for selecting desired constructs and/or transformants,and an origin of replication. The choice of vector systems usuallydepends upon several factors, including, but not limited to, the choiceof host cells, replication efficiency, selectability, inducibility, andthe ease of recovery.

Constructs of the invention containing IS481 or IS1001 nucleic acidmolecules can be propagated in a host cell. As used herein, the termhost cell is meant to include prokaryotes and eukaryotes such as yeast,plant and animal cells. Prokaryotic hosts may include E. coli,Salmonella tymphimurium, Serratia marcescens and Bacillus subtilis.Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichiapastoris, mammalian cells such as COS cells or Chinese hamster ovary(CHO) cells, insect cells, and plant cells such as Arabidopsis thalianaand Nicotiana tabacum. A construct of the invention can be introducedinto a host cell using any of the techniques commonly known to those ofordinary skill in the art. For example, calcium phosphate precipitation,electroporation, heat shock, lipofection, microinjection, andviral-mediated nucleic acid transfer are common methods for introducingnucleic acids into host cells. In addition, naked DNA can be delivereddirectly to cells (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in the present invention include oligonucleotidescapable of acting as a point of initiation of nucleic acid synthesiswithin IS481 or IS1001 nucleic acid sequences. A primer can be purifiedfrom a restriction digest by conventional methods, or it can be producedsynthetically. The primer is preferably single-stranded for maximumefficiency in amplification, but the primer can be double-stranded.Double-stranded primers are first denatured, i.e., treated to separatethe strands. One method of denaturing double-stranded nucleic acids isby heating.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T aquaticus, T lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

If the B. pertussis or B. parapertussis template nucleic acid isdouble-stranded, it is necessary to separate the two strands before itcan be used as a template in PCR. Strand separation can be accomplishedby any suitable denaturing method including physical, chemical orenzymatic means. One method of separating the nucleic acid strandsinvolves heating the nucleic acid until it is predominately denatured(e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). Theheating conditions necessary for denaturing template nucleic acid willdepend, e.g., on the buffer salt concentration and the length andnucleotide composition of the nucleic acids being denatured, buttypically range from about 90° C. to about 105° C. for a time dependingon features of the reaction such as temperature and the nucleic acidlength. Denaturation is typically performed for about 30 sec to 4 min.

If the double-stranded nucleic acid is denatured by heat, the reactionmixture is allowed to cool to a temperature that promotes annealing ofeach primer to its target sequence on the template nucleic acid. Thetemperature for annealing is usually from about 35° C. to about 65° C.Annealing times can be from about 10 secs to about 1 min. The reactionmixture is then adjusted to a temperature at which the activity of thepolymerase is promoted or optimized, i.e., a temperature sufficient forextension to occur from the annealed primer to generate productscomplementary to the template nucleic acid. The temperature should besufficient to synthesize an extension product from each primer that isannealed to a nucleic acid template, but should not be so high as todenature an extension product from its complementary template (e.g., thetemperature for extension generally ranges from about 40° to 80° C.).Extension times can be from about 10 secs to about 5 mins.

PCR assays can employ template nucleic acid such as DNA or RNA,including messenger RNA (mRNA). The template nucleic acid need not bepurified; it may be a minor fraction of a complex mixture, such as B.pertussis or B. parapertussis nucleic acid contained in human cells. DNAor RNA may be extracted from a biological sample such as nasopharyngealswabs, nasopharyngeal aspirates, and throat swabs by routine techniquessuch as those described in Diagnostic Molecular Microbiology: Principlesand Applications (Persing et al. (eds), 1993, American Society forMicrobiology, Washington D.C.). Template nucleic acids can be obtainedfrom any number of sources, such as plasmids, or natural sourcesincluding bacteria, yeast, viruses, organelles, or higher organisms suchas plants or animals.

The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH8.3), 1.5 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO). The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target nucleic acid molecule. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Fluorescent Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on the concept that when a donor anda corresponding acceptor fluorescent moiety are positioned within acertain distance of each other, energy transfer takes place between thetwo fluorescent moieties that can be visualized or otherwise detectedand/or quantitated. Two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the target nucleic acid sequence. Uponhybridization of the oligonucleotide probe to the amplification productat the appropriate positions, a FRET signal is generated. Hybridizationtemperatures can range from about 35° C. to about 65° C. for about 10seconds to about 1 minute.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system or afluorometer. Excitation to initiate energy transfer can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptorfluorescent moieties, “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radiative energytransfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Förster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LCT™-Red 640, LC™-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of thelinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm for the purpose of the present invention is the distance inAngstroms (Å) from the nucleotide base to the fluorescent moiety. Ingeneral, a linker arm is from about 10 to about 25 Å. The linker arm maybe of the kind described in WO 84/03285. WO 84/03285 also disclosesmethods for attaching linker arms to a particular nucleotide base, andalso for attaching fluorescent moieties to a linker arm.

An acceptor fluorescent moiety such as an LCT™-Red 640-NHS-ester can becombinated with C6-Phosphoramidites (available from ABI (Foster City,Calif.) or Glen Research (Sterling, Va.)) to produce, for example,LC-Red 640-Phosphoramidite. Frequently used linkers to couple a donorfluorescent moiety such as fluorescein to an oligonucleotide includethiourea linkers (FITC-derived, for example, fluorescein-CPG's from GlenResearch or ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPG's that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of B. pertussis and/or B. parapertussis

The present invention provides methods for detecting the presence orabsence of B. pertussis and/or B. parapertussis in a biological samplefrom an individual. The methods include performing at least one cyclingstep that first includes contacting the sample with a pair of IS481and/or IS1001 primers to produce an IS481 amplification product if B.pertussis is present in the sample, and/or an IS1001 amplificationproduct if B. parapertussis is present in the sample. Each of the IS481or IS1001 primers anneals to a target within or adjacent to a IS481 orIS1001 nucleic acid molecule, respectively, such that at least a portionof each amplification product contains nucleic acid sequencecorresponding to IS481 or IS1001, respectively. More importantly, theamplification product should contain the nucleic acid sequences that arecomplementary to the IS481 or IS1001 probes, respectively. Each cyclingstep further includes contacting the sample with a pair of IS481 and/orIS1001 probes. According to the invention, one member of each pair ofthe IS481 and IS1001 probes is labeled with a donor fluorescent moietyand the other is labeled with a corresponding acceptor fluorescentmoiety. The presence or absence of FRET between the donor fluorescentmoiety of the first IS481 or IS1001 probe and the corresponding acceptorfluorescent moiety of the second IS481 or IS1001 probe, respectively, isdetected upon hybridization of the probes to the respectiveamplification product. Multiple cycles of amplification andhybridization are performed, preferably in a thermocycler.

The methods of the invention can be performed individually to detecteither B. pertussis or B. parapertussis, but combining the primers andprobes in a single assay to detect the repetitive insertion molecules(IS481/IS1001) of B. pertussis and B. parapertussis provides a rapid andsensitive test that can distinguish between the species in a singlereaction. Representative biological samples that can be used inpracticing the methods of the invention include nasopharyngeal swabs,nasopharyngeal aspirates, throat swabs, or any biological specimen orswab containing ciliated respiratory epithelium that has the potentialto harbor Bordetella species. Biological samples are generally processed(e.g., by nucleic acid extraction methods known in the art) to releaseBordetella nucleic acid.

As used herein, “amplifying” refers to the process of synthesizingnucleic acid molecules that are complementary to one or both strands ofa template nucleic acid molecule (e.g., IS481 or IS1001 nucleic acidmolecules). Amplifying a nucleic acid molecule typically includesdenaturing the template nucleic acid, annealing primers to the templatenucleic acid at a temperature that is below the melting temperatures ofthe primers, and enzymatically elongating from the primers to generatean amplification product. Amplification typically requires the presenceof deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g.,Platinum® Taq) and an appropriate buffer and/or co-factors for optimalactivity of the polymerase enzyme (e.g., MgCl₂ and/or KCl).

If amplification of Bordetella nucleic acid occurs and an amplificationproduct is produced, the step of hybridizing results in a detectablesignal based upon FRET between the members of the pair of probes. Asused herein, “hybridizing” refers to the annealing of probes to anamplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the IS481 and IS1001 probes from therespective amplification product(s) can confirm the presence or absenceof B. pertussis and B. parapertussis in the sample, and can distinguishbetween B. pertussis and B. parapertussis. Alternatively, a Lightcycler™apparatus allows for multiple wavelengths to be measured simultaneously.Therefore, the second IS481 and IS1001 probe can be labeled withdifferent acceptor fluorescent moieties (e.g., LC-Red 640 and LC-Red705), thereby providing a method of distinguishing between B. pertussisand B. parapertussis based on differential FRET signals.

Generally, the presence of FRET indicates the presence of B. pertussisand/or B. parapertussis in the biological sample, and the absence ofFRET indicates the absence of B. pertussis and B. parapertussis in thebiological sample. Using the methods disclosed herein, detection of FRETwithin 40 cycles (e.g., within 30, 25, or 20 cycles) is indicative of aB. pertussis and/or B. parapertussis infection. A positive resultindicates the presence of nucleic acid from B. pertussis and/or B.parapertussis in the biological sample. In some cases, a positive resultwill be positive for both B. pertussis and B. parapertussis. A negativeresult indicates the absence of detectable DNA in the specimen submittedfor analysis, but does not negate the possibility of the organism'spresence in very small quantities. A negative result can occur wheninhibitory substances are present in the specimen (studies herein havedemonstrated 14% of nasopharyngeal specimens contain unknownPCR-inhibitory components). Inadequate specimen collection,transportation delays, inappropriate transportation conditions, or useof certain collection swabs (calcium alginate or aluminum shaft) are allconditions that can affect the success and/or accuracy of the testresult.

Methods of the invention also can be used for vaccine efficacy studiesor epidemiology studies of either or both B. pertussis and B.parapertussis. For example, an attenuated B. pertussis or B.parapertussis in a vaccine can be detected using the methods of theinvention during the time when bacteria is still present in anindividual. For such vaccine efficacy studies, the methods of theinvention can be used to determine, for example, the persistence of anattenuated strain of B. pertussis or B. parapertussis used in a vaccine,or can be performed in conjunction with an additional assay such as aserologic assay to monitor an individual's immune response to such avaccine. In addition, methods of the invention can be used todistinguish one B. pertussis or B. parapertussis strain from another forepidemiology studies of, for example, the origin or severity of anoutbreak of B. pertussis or B. parapertussis, respectively.

Methods of the invention are highly sensitive and highly specific. Thereal-time PCR method disclosed herein is far more sensitive than cultureand DFA and superior to the conventional PCR due to the ability todifferentiate between two species of Bordetella. The methods of theinvention do not require gel electrophoresis or Southern hybridization,making the methods described herein much more rapid than any Bordetelladetection method currently available. Rapid diagnosis leading totreatment with antibiotics can prevent potentially serious consequencesfrom Bordetella respiratory infections.

Within each thermocycler run, control samples are cycled as well.Positive control samples can amplify Bordetella nucleic acid controltemplate (other than the IS481 or IS1001 nucleic acid) using, forexample, control primers and control probes. Positive control samplescan also amplify, for example, a plasmid construct containing IS481and/or IS1001. Such a plasmid control can be amplified internally (e.g.,within each sample) or in a separate sample run side-by-side with thepatients' samples. The use of such controls can identify false-negativesdue, for example, to the inhibition of PCR observed with some samples.Each thermocycler run should also include a negative control that, forexample, lacks template DNA.

In an embodiment, the methods of the invention include steps to avoidcontamination. For example, an enzymatic method utilizing uracil-DNAglycosylase is described in U.S. Pat. Nos. 5,035,996, 5,683,896 and5,945,313 to reduce or eliminate contamination between one thermocyclerrun and the next. In addition, standard laboratory containment practicesand procedures are desirable when performing methods of the invention.Containment practices and procedures include, but are not limited to,separate work areas for different steps of a method, containment hoods,barrier filter pipette tips and dedicated air displacement pipettes.Consistent containment practices and procedures by personnel arenecessary for accuracy in a diagnostic laboratory handling clinicalsamples.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods of the invention. In one embodiment, aLightCycler™ instrument is used. A detailed description of theLightCycler™ System and real-time and on-line monitoring of PCR can befound at http://biochem.roche.com/lightcycler. The following patentapplications describe real-time PCR as used in the LightCycler™technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler™instrument is a rapid thermocycler combined with a microvolumefluorometer utilizing high quality optics. This rapid thermocyclingtechnique uses thin glass cuvettes as reaction vessels. Heating andcooling of the reaction chamber are controlled by alternating heated andambient air. Due to the low mass of air and the high ratio of surfacearea to volume of the cuvettes, very rapid temperature exchange ratescan be achieved within the LightCycler™ thermal chamber. Addition ofselected fluorescent dyes to the reaction components allows the PCR tobe monitored in real-time and on-line. Furthermore, the cuvettes serveas an optical element for signal collection (similar to glass fiberoptics), concentrating the signal at the tip of the cuvettes. The effectis efficient illumination and fluorescent monitoring of microvolumesamples.

The LightCycler™ carousel that houses the cuvettes can be removed fromthe instrument. Therefore, samples can be loaded outside of theinstrument (in a PCR Clean Room, for example). In addition, this featureallows for the sample carousel to be easily cleaned and sterilized. Thefluorimeter, as part of the LightCycler™ apparatus, houses the lightsource. The emitted light is filtered and focused by an epi-illuminationlens onto the top of the cuvettes. Fluorescent light emitted from thesample is then focused by the same lens, passed through a dichroicmirror, filtered appropriately, and focused onto data-collectingphotohybrids. The optical unit currently available in the LightCycler™instrument (Roche Molecular Biochemicals, Catalog No. 2 011 468)includes three band-pass filters (530 nm, 640 nm, and 710 nm), providingthree-color detection and several fluorescence acquisition options. Datacollection options include once per cycling step monitoring, fullycontinuous single-sample acquisition for melting curve analysis,continuous sampling (in which sampling frequency is dependent on samplenumber) and/or stepwise measurement of all samples after definedtemperature interval.

The LightCycler™ can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the cuvettes sequentially over the optical unit.The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

A common FRET technology format utilizes two hybridization probes. Eachprobe can be labeled with a different fluorescent moiety and aregenerally designed to hybridize in close proximity to each other in atarget DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler™ Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler™-Red 640 (LC™-Red 640) or LightCycler™-Red 705 (LC™-Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler™ instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of B. pertussis or B. parapertussisorganisms).

Another FRET technology format utilizes TaqMan® technology to detect thepresence or absence of an amplification product, and hence, the presenceor absence of B. pertussis or B. parapertussis. TaqMan® technologyutilizes one single-stranded hybridization probe labeled with twofluorescent moieties. When a first fluorescent moiety is excited withlight of a suitable wavelength, the absorbed energy is transferred to asecond fluorescent moiety according to the principles of FRET. Thesecond fluorescent moiety is generally a quencher molecule. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product) and isdegraded by the 5′ to 3′ exonuclease activity of the Taq Polymeraseduring the subsequent elongation phase. As a result, the excitedfluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.) uses TaqMan® technology, and issuitable for performing the methods described herein for detectingBordetella. Information on PCR amplification and detection using an ABIPRISM® 770 system can be found athttp://www.appliedbiosystems.com/products.

Yet another FRET technology format utilizes molecular beacon technologyto detect the presence or absence of an amplification product, andhence, the presence or absence of Bordetella. Molecular beacontechnology uses a hybridization probe labeled with a donor fluorescentmoiety and an acceptor fluorescent moiety. The acceptor fluorescentmoiety is generally a quencher, and the fluorescent labels are typicallylocated at each end of the probe. Molecular beacon technology uses aprobe oligonucleotide having sequences that permit secondary structureformation (e.g., a hairpin). As a result of secondary structureformation within the probe, both fluorescent moieties are in spatialproximity when the probe is in solution. After hybridization to thetarget nucleic acids (i.e., amplification products), the secondarystructure of the probe is disrupted and the fluorescent moieties becomeseparated from one another such that after excitation with light of asuitable wavelength, the emission of the first fluorescent moiety can bedetected.

As an alternative to detection using FRET technology, an amplificationproduct can be detected using a nucleic acid binding dye such as afluorescent DNA binding dye (e.g., SYBRGreenI® or SYBRGold® (MolecularProbes)). Upon interaction with the double-stranded nucleic acid, suchnucleic acid binding dyes emit a fluorescence signal after excitationwith light at a suitable wavelength. A nucleic acid binding dye such asa nucleic acid intercalating dye also can be used. When nucleic acidbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

It is understood that the present invention is not limited by theconfiguration of one or more commercially available instruments.

Articles of Manufacture

The invention further provides for articles of manufacture to detect B.pertussis and/or B. parapertussis. An article of manufacture accordingto the present invention can include primers and probes used to detectB. pertussis or B. parapertussis, together with suitable packagingmaterials. Representative primers and probes for detection of B.pertussis are capable of hybridizing to IS481 nucleic acid molecules.Representative primers and probes for detection of B. parapertussis arecapable of hybridizing to IS1001 nucleic acid molecules. Methods ofdesigning primers and probes are disclosed herein, and representativeexamples of primers and probes that amplify and differentially detect toB. pertussis and B. parapertussis nucleic acid molecules are providedherein.

Articles of manufacture of the invention can also include one or morefluorescent moieties for labeling the probes or, alternatively, theprobes supplied with the kit can be labeled. For example, an article ofmanufacture may include a donor fluorescent moiety for labeling one ofthe IS481 or IS1001 probes and a corresponding acceptor fluorescentmoiety for labeling the other IS481 or IS1001 probe, respectively.Examples of suitable FRET donor fluorescent moieties and correspondingacceptor fluorescent moieties are provided herein.

Articles of manufacture of the invention also can contain a packageinsert or package label having instructions thereon for using the IS481primers and probes to detect the presence or absence of B. pertussis ina biological sample and, likewise, using the IS1001 primers and probesto detect the presence or absence of B. parapertussis in a sample. Sucha package insert may further contain instructions thereon for usingIS481 and IS1001 probes to distinguish between B. pertussis and B.parapertussis within the same biological sample. Articles of manufacturemay additionally include reagents for carrying out the methods disclosedherein (e.g., buffers, polymerase enzymes, co-factors, or agents toprevent contamination). Such reagents may be specific for one of thecommercially available instruments described herein.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 DNA Extraction and Bordetella LightCycler™ Assay #1

Recovery of Bordetella from nasopharyngeal swabs was achieved byswabbing the nasopharynx with a nylon swab having an aluminum shaft andtransported in media. Upon arrival at Mayo, the swab was placed in 500μl Sample Buffer (Reagent A) in a 1.5 ml microcentrifuge tube and storedat 2-8° C. 200 μl of the specimen in Sample Buffer was used for DNAextraction and the remainder was saved at −70° C. for future use. SampleBuffer was added to nasopharyngeal aspirates to bring the volume up to500 μl.

The DNA sample was taken into a ‘PCR Set-Up Room’ and 200 μl of thesample was transferred into 2.0 ml microcentrifuge tubes. DNA extractionwas performed in an ‘Extraction PCR Workstation’ using an Isoquick DNAExtraction Kit (ORCA Research, Inc.; Bothell, Wash.; Catalog #217539).Pellet Paint™ NF Co-Precipitant (Novagen; Madison, Wis.; Catalog#70748-3) was used in all extractions. For diagnostic labs runningmultiple tests on the same biological sample, refer to ‘Nucleic AcidProcedures Shared among Molecular Microbiology Tests’ provided with theIsoquick DNA Extraction kit.

Alternatively, DNA was prepared from a sample by boiling andcentrifugation. Briefly, the swab was rinsed with 200 μl of water thatwas collected in a 2 ml screw cap tube. The tube was centrifuged at13,000×g for 1 min and the supernatant was removed. The pellet wasresuspended in 100 μl of RNase-free water and boiled at 100° C. for 10min. The tube was centrifuged at 13,000×g for 1 min and the supernatantcollected.

In a ‘PCR Clean Room’, the frozen B. pertussis and B. parapertussisLightCycler™ PCR master-mixes were thawed, vortexed briefly andcentrifuged for 1 minute at 20,800×g. If prepared separately, the B.pertussis and B. parapertussis master mixes were combined 1:1 in a 1.5ml Eppendorf tube and mixed. The amount of time the reagents were leftat room temperature was minimized. The LightCycler™ carousel was loadedwith two cuvettes representing positive controls, an appropriate numberof negative controls, and the remainder with patient's samples. 15 μl ofthe combined Bordetella PCR master-mix was added to each cuvette using arepeat pipettor.

The cuvettes containing the Bordetella PCR master-mix were transferredto a ‘Target Loading PCR Workstation’ and 5 μA of the sample supernatantwas carefully removed and added to the 15 μA of Bordetella PCRmaster-mix in each LightCycler™ cuvette. The cuvettes were capped. Thecarousel was transported to a ‘LightCycler™ Area’ and was centrifuged inthe LightCycler™ carousel centrifuge. The carousel was placed in theLightCycler™ apparatus and the Bordetella LightCycler™ program was run.Samples underwent 40 cycles of: denaturation at about 95° C. immediatelyfollowed by primers annealing to the template nucleic acid for about 20secs at about 60° C., and elongation of the newly-synthesized strands atabout 72° C. for about 14 secs. During the run, the specimen names wereentered and typed into the LightCycler™ software sample table. The runwas complete in about one hour.

After completion of the run, the cuvettes were removed from the carouselwith a cuvette extruder or by turning the carousel upside down andgently loosening the cuvettes until they fell into a collection bucket.The carousel was decontaminated in DNA-OFF (Daigger; Vernon Hills, Ill.;Cat. # HX12982) for 1 min, rinsed with de-ionized water and air driedbefore being returned to the ‘PCR Clean Room’.

Extreme care was taken to avoid all contact of sample or sample extractscontaining Bordetella DNA with any solutions or portion of theLightCycler™ apparatus prior to PCR amplification. False-positivereactions can occur due to cross-contamination fromBordetella-containing samples. For these reasons, the use of at leastthree separate areas for sample preparation and LightCycler™ setup arerecommended: an area for PCR mix preparation (e.g., a ‘PCR Clean Room’),an area for specimen processing and setting-up the PCR reactions (e.g.,an ‘Extraction PCR Workstation’ or a ‘Target Loading PCR Workstation’),and an area dedicated to the actual amplification reactions (e.g., a‘LightCycler™ Area’). Dedicated pipettes and barrier filter pipette tipscan be used with all air displacement pipettes and careful pipetting canminimize any cross-contamination events.

Example 2 Primers and Probes

Primers (0.2 μM medium scale synthesis) were ordered from the MayoMolecular Biology Core Facility (Rochester, Minn.). Primers were drieddown at 60° C. with vacuum (22 psi), and resuspended in 500 μl to 1 mlRNase-free water. Primers were adjusted to 50 μM by measuring the A₂₆₀of a 1/100 dilution (198 μA water+2 μA, DF=100). The concentration wasestimated by the following formula: [DF X A₂₆₀X 100/number of bases=μM].The concentration was adjusted to 50 μM by adding water using thefollowing formula: [((μM found/50)×μl remaining)−μl remaining=water toadd]. Primers were mixed (1:1) to make a stock solution containing 25 μMof each primer and stored at −20° C.

Probes were obtained from Idaho Technologies(http://www.idahotech.com/itbiochem/index.html). The probes weresuspended in 1× TE buffer supplied with the probes to a finalconcentration of 20 μM.

The A₂₆₀ and A₄₉₄ of the fluorescein-labeled probe were measured. Theextinction coefficient (e₂₆₀) of the fluorescein-labeled probe wascalculated using nearest neighbor values. The LightCycler™ Probe QC, anExcel spreadsheet, was used to calculate the extinction coefficients andratios.

The dye-oligonucleotide ratio was determined. The ratio should bebetween 0.8 and 1.2, which indicates that there is one dye moleculepresent for every oligonucleotide molecule. Probes were diluted 1/20 in0.5× TE buffer (pH 8.3) to determine this ratio. The extinctioncoefficient of fluorescein is very sensitive to pH. [DyeμM=(A₄₉₄/68,000)]. [Oligo μM=[A₂₆₀−(A₄₉₄×12,000/68,000)]/e₂₆₀×DF×10⁶].

The A₂₆₀ and A₆₂₂ of the LC-Red 640-labeled oligonucleotide weremeasured and the predicted extinction coefficient (e₂₆₀) was calculatedusing nearest neighbor values. [Dye μM=(A₆₂₂/110,000)]. [OligoμM=[A₂₆₀−(A₆₂₂×31,000/110,000)]/e₂₆₀×DF×10⁶].

TABLE 1 Primers and probes for detection of B. pertussis Product SEQ IDType Size (bp) Name Sequences (5′→3′) NO: Primer 234 BP IS694ccagttcctcaaggacgc 1 Primer 234 BP IS905 gagttctggtaggtgtgag 2 cgtaProbe BP IS F caccgctttacccgacctt 3 accgcccac Probe BP IS Rgaccaatggcaaggccgaa 4 cgcttcatc F = fluorescein-labeled probeoligonucleotide; R = LC-Red 640-labeled probe oligonucleotide

TABLE 2 Primers and probes for detection of B. parapertussis Product SEQID Type Size (bp) Name Sequences (5′→3′) NO: Primer 200 BPP A375ggcgatatcaacgggtga 5 Primer 200 BPP A556 cagggcaaactcgtccatc 6 Probe BPPF gttcttcgaactgggttgg 7 catac Probe BPP R gtcaagacgctggacaagg 8 ctc F= fluorescein-labeled probe oligonucleotide; R = LC-Red 640-labeledprobe oligonucleotide

Example 3 Bordetella LightCycler™ Assay #1

LightCycler™ PCR master-mixes were prepared in the ‘PCR Clean Room’.This room was designed with positive airflow and is operated to minimizecontamination with nucleic acid from specimens or positive controls.Disposable gowns and gloves were worn at all times.

LightCycler™ PCR mix was prepared according to the following chart. B.pertussis IS481 PCR mix and B. parapertussis IS1001 PCR mix werealiquoted into separate 2.0 ml screw-capped microcentrifuge tubes andstored at −70° C. for up to 6 mo. All reagents were thawed, gentlyvortexed and quick spun prior to use (except for Platinum® Taq, whichwas only quick spun). The LightCycler™ PCR mix was prepared as soon asthe reagents were thawed.

LightCycler ™ PCR Master Mix - B. pertussis IS481 Number of reactions =>50 Target volume => 5 Stock Mix Ingredient Stock Conc. Mix Conc. (μl)Water 456.5 MgCl2 50 mM 4 mM 80 10X Platinum ® buffer 10 x 1 X 100Primers 25 μM 0.75 μM 30 Platinum ® Taq 5 U/μl 0.025 U/μl 5 dNTP plus 10mM 0.2 mM 20 BSA 2 % 0.025 % 12.5 HK-UNG 10 % 0.2 % 20 BP IS F probe 20μM 0.2 μM 10.0 BP IS R probe 20 μM 0.3 μM 15.0 dNTP plus = 1X each ofdATP, dCTP, and dGTP, 3X of dUTP

LightCycler ™ Hybridization Master Mix - B. parapertussis IS1001 Numberof reactions => 50 Target volume => 5 Stock Mix Ingredient Stock Conc.Mix Conc. (μl) Water 447.5 MgCl2 50 mM 4 mM 80 10X Platinum ® buffer 10x 1 X 100 Primers 25 μM 0.75 μM 30 DMSO 100 % 1 % 10 Platinum ® Taq 5U/μl 0.025 U/μl 5 dNTP plus 10 mM 0.2 mM 20 BSA 2 % 0.025 % 12.5 HK-UNG10 % 0.2 % 20 BPP F probe 20 μM 0.2 μM 10.0 BPP R probe 20 μM 0.3 μM15.0 dNTP plus = 1X each of dATP, dCTP, and dGTP, 3X of dUTP

Alternatively, a single master mix can be generated to detect either orboth B. pertussis or B. parapertussis in a biological sample.

LightCycler ™ Hybridization Master Mix B. pertussis IS481 and B.parapertussis IS1001 Number of reactions => 50 Target volume => 5 StockMix Ingredient Stock Conc. Mix Conc. (μl) Water 421.5 MgCl₂ 50 mM 4 mM80 10X Platinum ® buffer 10 x 1 X 100 Primers (B. pertussis) 25 μM 0.5μM 20 Primers (B. parapertussis) 25 μM 0.5 μM 20 Platinum ® Taq 5 U/μl0.03 U/μl 6 dNTP plus 10 mM 0.2 mM 20 BSA 2 % 0.025 % 12.5 HK-UNG 10 %0.2 % 20 BP F probe 20 μM 0.2 μM 10 BP R probe 20 μM 0.3 μM 15 BPP Fprobe 20 μM 0.2 μM 10 BPP R probe 20 μM 0.3 μM 15

Example 4 Quality Control

A positive control of both B. pertussis (ATCC #9797) and B.parapertussis (ATCC#15311) were extracted and processed through theLightCycler™ detection in each clinical run. A melting curve analysiswas used to differentiate the two organisms. If amplification of thepositive control was not detected within 4 cycles of the expected numberof cycles for detection of positive controls, or does not amplify, therun was repeated.

A fresh culture of the ATCC strains of B. pertussis and B. parapertussiswere grown on charcoal agar at 37° C. in a CO₂ incubator. Severalcolonies were resuspended in sterile saline and adjusted to a MacFarlandstandard of 0.5 (ca.1.5×10⁸ organisms/ml) using the Vitek Colorimeter(85% T±2). A 10-fold dilution series was prepared using molecular gradewater (50 μl dilution into 450 μl water). Recovery studies wereperformed by adding 20 μl of each dilution of the series to SampleBuffer and extracting the DNA. The optimal concentration of B. pertussisand B. parapertussis was determined and a stock solution of theappropriately diluted culture was made in molecular grade water andstored at 4° C.

A positive control was generated by cloning IS481 or IS1001 nucleic acidmolecules into a vector using the Invitrogen TOPO TA Cloning kit (Cat.#K4500-01). The 234 by PCR amplicon of B. pertussis and the 200 by PCRamplicon of B. parapertussis were each inserted into a plasmid vector(pCR 2.1-TOPO). The recombinant vector was transformed into chemicallycompetent E. coli and grown overnight on a LB agar plate containing 50μg/ml of kanamycin. The white colonies containing the confirmedrecombinant plasmid were grown overnight in LB broth containingkanamycin and purified with the Promega Wizard Plus MiniPrep DNApurification system (Cat. #A7500). The stock concentration of thepositive plasmid control was determined in molecules/μl. A ten-foldserial dilution was prepared using 20 μl of the suspension and 180 μl ofsterile RNAse-free water. This dilution series was carried through untilno amplification product was detected. Each dilution was tested with theBordetella LightCycler™ assay and the optimal positive control dilutionwas determined. A working solution of 1.0 ml of this dilution wasprepared and stored at 4° C.

Alternatively, the positive control was extracted from a culture (20 μAcontrol plus 180 μA Isoquick Sample Buffer) and processed in parallelwith the clinical specimens to provide a consistent means of monitoringassay performance. Negative controls were included in each clinical run.Negative controls consisted of 5-10% of the batch and were interspersedin the LightCycler™ apparatus with patient samples. These controlstested for hybridization mix contamination and specimen-to-specimencarryover contamination. If a negative control(s) yielded a positivereaction, extraction reagents were replaced and the samples and controlsfrom the run in question were re-extracted.

Isoquick solution Sample Buffer A was extracted and used as a negativecontrol. This was to confirm that extraction reagents were notcontaminated with previously amplified product. Alternatively, 5 μA ofwater was added directly to the Master Mix and amplified as a negativecontrol. All specimens (patient's and controls) were handled usingUniversal Precautions. Sterile gloves were worn when handling samplesand performing all procedures. Gloves were changed frequently.

dUTP incorporation and uracil N-glycosylase treatment with athermolabile UNG (Epicentre Technologies; Madison, Wis.; Catalog#HU5910K) were used to prevent amplicon carryover in the LightCycler™assays described herein. Although not required, the routineimplementation of these precautions diminishes the risk offalse-positive results. False-positive results have been a significantand often cited problem in many laboratories using PCR techniques andcan seriously compromise the reliability of testing performed in aclinical environment.

Example 5 Interpreting and Reporting Results

A clinical specimen that displayed a melting temperature of 75°±2° C.was interpreted as positive for B. pertussis and/or a meltingtemperature of 64°±2° C. was interpreted as positive for B.parapertussis DNA.

The B. pertussis IS481 assay is specific for B. pertussis and a positivesignal is reported as B. pertussis. Although the primers and probes arespecific for the insertion sequence of B. pertussis IS481,cross-reactivity with B. holmesii can occur with B. pertussis IS481 PCRassays. Cephalexin, the antibiotic widely used in culture media, has aninhibitory effect on B. holmesii. In one evaluation, B. holmesiipositivity rate in nasopharyngeal specimens was 0.29%. The clinicalsignificance of B. holmesii has yet to be determined although it hasbeen associated with septicemia, respiratory failure and symptomssimilar to B. pertussis infection (i.e., cough).

The B. parapertussis IS1001 assay is specific for B. parapertussisspecies and a positive signal is reported as B. parapertussis. Becausethe primers and probes are specific for B. parapertussis, and nocross-reactions have been observed with these reagents, a positive testwill provide results of the specific nucleic acid. Therefore, positiveresults can be reported as B. parapertussis.

A clinical specimen or control with no melting curve above baselineshould be interpreted as negative for the presence of B. pertussis or B.parapertussis DNA. Results are strictly qualitative. A negative resultdoes not negate the presence of the organism or active disease. Testresults should be used as an aid in diagnosis and not be considered astand-alone diagnostic test. A single assay should not be used as theonly criteria to form a clinical conclusion, but results should becorrelated with serologic tests, patient symptoms, and clinicalpresentation.

Example 6 Method Validation

The LightCycler™ PCR assay for detection of B. pertussis and/or B.parapertussis was compared to culture/DFA, to conventional PCR of theIS481 gene of B. pertussis, and to a LightCycler™ PCR assay fordetection of the pertussis toxin gene (PTG). A combined gold standardwas used to compare the LightCycler™ PCR assay to the other detectionmethods. This gold standard is defined as ≧1 positive result in anycombination of results from culture/DFA, PTG, and conventional PCR.

Compared to culture/DFA and a LightCycler™ PCR assay for detection ofPTG, the LightCycler™ PCR assay for detection of B. pertussis and B.parapertussis using IS481 and IS1001, respectively, was 100% sensitiveand 72% specific (p<0.0001 using a kappa statistic). The positivepredictive value (ppv) and the negative predictive value (npv) were 30%and 100%, respectively. Compared to the LightCycler™ PCR assay,conventional PCR of the IS481 gene had a sensitivity of 96%, specificityof 82%, ppv of 83%, and an npv of 95% (p=0.0654). Compared toLightCycler™ PCR and PTG LightCycler™ PCR, culture/DFA had a sensitivityof 25%, specificity of 100%, ppv of 100%, and an npv of 70% (p<0.0001).Compared to LightCycler™ PCR and culture/DFA, the PTG LightCycler™ assayhad a sensitivity of 28%, specificity of 100%, ppv of 100%, and an npvof 71% (p<0.0001). Using dilutions of well-characterized American TypeCulture Collection (ATCC) and CDC positive controls, the sensitivity ofthe IS481/IS1001 assay was 1 organism/μl for both the detection of B.pertussis and B. parapertussis.

A low level positive control of B. pertussis and B. parapertussis wasrun multiple times within a run, two times within a day and on threeconsecutive days. The variability was determined to be in the acceptablerange of ±4 cycles. The analytical detection limit of the LightCycler™PCR assay, using dilutions of a McFarland 0.5 standard of freshcultures, was 1 organism per reaction. The average number of templatesper organism was 80 in B. pertussis and 20 in B. parapertussis.

Ninety-two Isoquick extracted nasopharyngeal samples were spiked with B.pertussis and B. parapertussis and tested for the presence ofinhibitors. 13 samples did not amplify under such conditions, giving aninhibition rate of 14%. The choice of transport swab and medium mayaffect inhibition (e.g., calcium alginate swabs, cotton swabs, andaluminum shaft swabs may be inhibitory to PCR).

Example 7 Bordetella LightCycler Assay #2 with Recovery Template

200 μl sterile water was added to an original tube containing anasopharyngeal swab, and the tube was vortexed well. 200 μl of the swabmaterial was transferred to a screw-capped tube containing 4 μl ofrecovery template (5×10³ targets/μl), and the tube was capped and mixedbriefly. Recovery template is modified template nucleic acid that isco-amplified in the same tube by the same set of primers used to amplifytemplate nucleic acid. Recovery template, however, uses different probesfor detection. The probes that hybridize to the recovery template(apoE-F and apoE 705) are labeled with fluorescein and LC-Red 705 sothat amplification of the recovery template is measured on a differentchannel than the channel used to measure amplification of the template.Recovery template can be used to identify samples containing aninhibitory component.

100 μl of STAR Buffer (Roche Molecular Diagnostics, Indianapolis, Ind.)was placed into a MagNA Pure sample cartridge. 100 μl of the swab sample(containing recovery template at a final concentration of 1×10²targets/μl) was transferred into extraction wells, and the DNA extractedusing MagNA Pure and the LightCycler Total Nucleic Acid Isolation kit.

The remaining 100 μl of the swab sample was placed in a 95° C. (±5° C.)heat block for 10 min. The tube was centrifuged for 3 min at 20,000×g topellet any particulate material.

Example 8 Control Samples

Several types of positive controls were used. For a positive control ofeach organism, B. pertussis (ATCC Accession No. 9797) or B.parapertussis (ATCC Accession No. 15311) were grown on charcoal agar anddiluted to a McFarland 0.5 (1.5×10⁸ cells/ml). The cultures were furtherdiluted to a working concentration of 1.5×10⁶ cells/ml and used ascontrols for extraction and amplification reactions of template from theboiled lysate procedure and from the MagNA Pure extraction. A positivecontrol corresponding to each Bordetella organism included 198 μlsterile water+2 μl organism control (1.5×10⁶ cells/μl)+4 μl recoverytemplate (5×10³ targets/μl).

In addition to the organism controls, a plasmid containing Bordetellasequences was used as a positive control. The plasmid contains sequencesfrom both IS481 (B. pertussis) and IS1001 (B. parapertussis) (RocheMolecular Diagnostics). A positive extraction control corresponding tothe plasmid included 198 μl sterile water+2 μl plasmid (2×10³targets/μl)+4 μl recovery template (5×10³ targets/μl).

A negative control included 200 μl sterile water+4 μl recovery template(5×10³ targets/μl).

Example 9 Bordetella LightCycler Assay #2

Tables 3 and 4 describe the sequences of the primers and probes used fordetection of B. pertussis and B. parapertussis, respectively.

TABLE 3 SEQ ID Type Name Sequences 5′→3′ NO: Primer BP IS1ccagttcctcaaggacgc 1 Primer BP IS2 gagttctggtaggtgtgagcgta 2 ProbeBP-F(WT) caccgctttacccgaccttaccgccca 3 c Probe BP853mm29gaccaatggcaaggctcgaacgcttca 11 tc

TABLE 4 Type Name Sequences (5′→3′) SEQ ID NO: Primer BPP IS1ggcgatatcaacgggtga 5 Primer BPP IS2 cagggcaaactcgtccatc 6 Probe VPP03ggttggcataccgtcaaga 12 Probe VPP04 gctggacaaggctcg 13

The components of the Bordetella LightCycler Assay #2 reaction mix areas follows.

Reagent Volume Water 11 μl Primer/Probe Mix^(a)  2 μl FastStart DNAMaster Hybridization Probes^(b)  2 μl Total 15 μl ^(a)primer/probe mixincludes: 2.5 mM MgCl₂; 0.3 μM each BP IS1 and BPP IS1; 0.5 μM each BPIS2 and BPP IS2; 0.2 μM each BP-F, VPP03, apoE-F, and apoE 705; and 0.4μM each BP853mm29 and VPP04. ^(b)magnesium is present in the FastStartDNA Master Hybridization Probe solution at a concentration of 1 mM.

As an alternative to adding the recovery template to the sample prior toextraction, recovery template can be added to the PCR reaction mix at afinal concentration of 5×10³ targets/μl.

5 μl from the boiled lysate, the MagNA Pure extraction, or the positiveor negative control samples, was mixed with 15 μl of the BordetellaLightCycler PCR reaction mix described above, placed in a LightCyclercarousel, and amplified as follows.

A. Initial 95° C. 10 min B. PCR 45 cycles 95° C. 10 sec 55° C. 15 secSingle signal 72° C. 15 sec C. Melt 95° C. 0 sec 20°/sec 59° C. 20 sec20°/sec 45° C. 20 sec 0.2°/sec 85° C. 0 sec 0.1°/sec Continuous SignalD. Melt 2 95° C. 0 sec 20°/sec 59° C. 20 sec 20°/sec 45° C. 20 sec0.2°/sec 85° C. 0 sec 0.2°/sec Continuous Signal E. Cool 40° C. 10 sec

Results of the experiments are shown in Table 5. The addition of therecovery template to the specimens prior to nucleic acid extraction didnot inhibit the extraction or the amplification of a product. A samplewas considered to contain an inhibitory component if the recoverytemplate amplification product was not generated in the absence oftarget template.

TABLE 5 Bordetella LightCycler Assay #2 Bordetella MagNA Pure Boil (w/LightCycler (w/ recovery recovery Sample Assay #1 MagNA Pure template)Boil template) 1 − − + − + 2 + B. pert − B. pert − 3 + B. pert + B.pert + 4 − − + − + 5 − − + − + 6 − − + − + 7 + B. para + B. para + 8 +B. pert + B. pert − 9 + B. pert + B. pert − 10 − − + − + 11 − − + − +12 + B. pert + B. pert + 13 − − + − + 14 + B. pert + B. pert + 15 − − +− + 16 − − + − + 17 − − + − + 18 + B. pert + B. pert + 19 − − + − + 20 +B. pert + B. pert + 21 − − + − + 22 + B. pert − B. pert − 23 + B. pert +B. pert + 24 + B. pert + B. pert + 25 − − + − + 26 − − + − + 27 − − +− + 28 − − + − + 29 + B. para + B. para + 30 + B. pert + B. pert +

Example 10 Specificity of Bordetella LightCycler Assay #2

Experiments were performed to determine if the Bordetella primers andprobes cross-reacted with nucleic from similar organisms or fromorganisms commonly found in the specimens tested.

5 μl from the boiled lysate or from the MagNA Pure extraction was addedto 15 μl of the PCR reaction mix described above in Example 9. Sampleswere placed into a LightCycler carousel and cycled as described above inExample 9. Nucleic acid had previously been shown to be amplifiable inthe bacterial organisms listed in Table 6 using 16S rRNA amplificationby conventional or LightCycler PCR assays. 2×10³ targets/μl of thepositive control plasmid described above in Example 8 was used.

TABLE 6 Organism Result Organism Result Pseudomonas aeruginosa −Morganella species − Chlamydia pneumoniae − Proteus vulgaris −Klebsiella pneumoniae − Mycoplasma pneumonia − Escherichia coli −Campylobacter jejuni − Haemophilus influenza − M. catarrhalis −Aeromonas species − Human DNA − Staphylococcus aureus − Bordetellapertussis + Legionella jordanis − Legionella pneumophila − S.maltophilia − Bordetella bronchioseptica − Klebsiella oxytoca −Neisseria meningitidis − Pseudomonas cepacia − Bordetella holmesii +Staphylococcus − Acinetobacter species − epidermidis Neisseriagonorrhoeae − Proteus mirabilis − Pseudomonas − Corynebacterium −fluorescens diphtheriae C. pseudodiptheriae − Bordetella parapertussis +

Other than the B. pertussis and B. parapertussis positive controls andthe closely related B. holmesii, which was previously detected with theBordetella Lightcycler Assay #1, none of the organisms shown in Table 6cross-reacted with the Bordetella primers and probes used in theBordetella LightCycler Assay #2.

Example 11 Diagnostic Sensitivity of the Bordetella LightCycler Assay #2

Experiments were performed to determine the sensitivity and specificityof the Bordetella LightCycler Assay #2 compared to culture and otheramplification methods. 110 nasopharyngeal swabs from patient samplessent in for Bordetella testing were examined using a culture method, anda portion of those samples were analyzed by a conventional PCR methodand the Bordetella LightCycler Assays #1 and #2 disclosed herein. Thenasopharyngeal swabs were initially cultured on charcoal agar platescontaining cephalexin and blood agar, incubated at 37° C. and examineddaily for 5 days for the presence of B. pertussis and/or B.parapertussis.

After swiping on agar plates, the nasopharyngeal swabs were swished in atube containing 500 μl sterile water. 200 μl was removed from the tube(n=35) and processed by boiling at 100° C. for 10 min. After boiling,the sample was centrifuged for 1 minute at 20,800×g. The DNA present in200 μl of the remaining nasopharyngeal swab sample was extracted usingthe Isoquick Nucleic Acid Extraction Kit (ORCA Research, Inc., Bothell,Wash.). These samples were stored at −20° C. for up to 48 months.

Using 99 of the 110 samples described above, 5 μl of the boiled lysateor the Isoquick extraction eluate was added to 15 μl of the PCR ReactionMix described above in Example 3. The samples were analyzed by theBordetella LightCycler Assay described above in Example 1 (BordetellaLightCycler Assay #1).

Another 5 μl of the boiled lysate or the IsoQick extraction eluate wasadded to 15 μl of the PCR Reaction Mix described above in Example 8. Thesamples were analyzed by the Bordetella LightCycler Assay describedabove in Example 8 (Bordetella LightCycler Assay #2).

A conventional PCR assay was used and amplified IS481 nucleic acidsequences. Following amplification by PCR, the sample waselectrophoresed on an agarose gel, Southern blotted, and detected byenzyme chemiluminescence (Amersham Corporation, Arlington Heights,Ill.). The conventional PCR protocol for detecting B. pertussis hadapproximately a 2-5 day turnaround time.

Results of culture methods versus the Bordetella LightCycler Assay #2are shown in Table 7. Results of experiments comparing BordetellaLightCycler Assay #1, Bordetella LightCycler Assay #2, and theconventional PCR assay are shown in Table 8.

TABLE 7 Culture B. pertussis B. parapertussis Negative Total BordetellaB. pertussis 18 0 23 41 LightCycler B. parapertussis 0 4 1  5 Assay #2Negative 0 0 59 59 Total 18 4 83 105* *The 5 samples unaccounted forwere considered indeterminate since recovery template was not detected.

TABLE 8 Bordetella LightCycler Assay #1 B. pertussis B. parapertussisNegative Total Bordetella B. pertussis 38 0 3 41 LightCycler B.parapertussis 0 2 1 3 Assay #2 Negative 9 2 44 55 TOTAL 47 4 48 99

Five samples were found to inhibit PCR amplification using theBordetella Lightcycler Assay #2 based upon a lack of detection of therecovery template. The correlation between the Bordetella LightCyclerAssay #1 and the Bordetella LightCycler Assay #2 was good. The integrityof some of the samples was questionable, with nucleic acid samplesarchived for a longer period of time sometimes resulting in a negativeresult.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for detecting the presence or absence of Bordetellaparapertussis in a biological sample from an individual, said methodcomprising: performing at least one cycling step, wherein a cycling stepcomprises an amplifying step and a hybridizing step, wherein saidamplifying step comprises contacting said sample with a pair of IS1001primers to produce an IS1001 amplification product if a B. parapertussisIS1001 nucleic acid molecule is present in said sample, wherein saidpair of IS1001 primers comprises a first IS1001 primer and a secondIS1001 primer, wherein said first IS1001 primer consists of the sequence5′-GGC GAT ATC AAC GGG TGA-3′ (SEQ ID NO:5) and said second IS1001primer consists of the sequence 5′-CAG GGC AAA CTC GTC CAT C-3′ (SEQ IDNO:6), wherein said hybridizing step comprises contacting saidbiological sample with a pair of IS1001 probes, wherein said pair ofIS1001 probes comprises a first IS1001 probe and a second IS1001 probe,wherein said first IS1001 probe consists of the sequence 5′-GGT TGG CATACC GTC AAG A-3′ (SEQ ID NO:12) and said second IS1001 probe consists ofthe sequence 5′-GCT GGA CAA GGC TCG-3′ (SEQ ID NO:13), wherein themembers of said pair of IS1001 probes hybridize within no more than fivenucleotides of each other, wherein a first IS1001 probe of said pair ofIS1001 probes is labeled with a donor fluorescent moiety and a secondIS1001 probe of said pair of IS1001 probes is labeled with acorresponding acceptor fluorescent moiety; and detecting the presence orabsence of FRET between said donor fluorescent moiety of said firstIS1001 probe and said corresponding acceptor fluorescent moiety of saidsecond IS1001 probe, wherein the presence of FRET is indicative of thepresence of B. parapertussis in said biological sample, and wherein theabsence of FRET is indicative of the absence of B. parapertussis in saidbiological sample.
 2. The method of claim 1, wherein the members of saidpair of IS1001 probes hybridize within no more than two nucleotides ofeach other.
 3. The method of claim 1, wherein the members of said pairof IS1001 probes hybridize within no more than one nucleotide of eachother.
 4. The method of claim 1, wherein said donor fluorescent moietyis fluorescein.
 5. The method of claim 1, wherein said acceptorfluorescent moiety is selected from the group consisting of LC™-Red 640,LC™-Red 705, Cy5, and Cy5.5.
 6. The method of claim 1, wherein saiddetecting step comprises exciting said biological sample at a wavelengthabsorbed by said donor fluorescent moiety and visualizing and/ormeasuring the wavelength emitted by said acceptor fluorescent moiety. 7.The method of claim 1, wherein said detecting comprises quantitatingsaid FRET.
 8. The method of claim 1, wherein said detecting step isperformed after each cycling step.
 9. The method of claim 1, whereinsaid detecting step is performed in real-time.
 10. The method of claim1, wherein the presence of said FRET within 40 cycling steps isindicative of the presence of a B. parapertussis infection in saidindividual.
 11. The method of claim 1, wherein the presence of said FRETwithin 30 cycling steps is indicative of the presence of a B.parapertussis infection in said individual.
 12. The method of claim 1,wherein the presence of said FRET within 25 cycling steps is indicativeof the presence of a B. parapertussis infection in said individual. 13.The method of claim 1, further comprising preventing amplification of acontaminant nucleic acid.
 14. The method of claim 13, wherein saidpreventing comprises performing said amplifying step in the presence ofuracil.
 15. The method of claim 14, wherein said preventing furthercomprises treating said biological sample with uracil-DNA glycosylaseprior to a first amplifying step.
 16. The method of claim 1, whereinsaid biological sample is selected from the group consisting ofnasopharyngeal swabs, nasopharyngeal aspirates, and throat swabs. 17.The method of claim 1, wherein said cycling step is performed on acontrol sample.
 18. The method of claim 17, wherein said control samplecomprises said portion of said IS1001 nucleic acid molecule.
 19. Themethod of claim 1, wherein said cycling step uses a pair of controlprimers and a pair of control probes, wherein said control primers andsaid control probes are other than said IS1001 primers and IS1001probes, respectively, wherein a control amplification product isproduced if control template is present in said sample, wherein saidcontrol probes hybridize to said control amplification product.
 20. Amethod for detecting the presence or absence of B. pertussis in abiological sample from an individual, said method comprising: performingat least one cycling step, wherein a cycling step comprises anamplifying step and a dye-binding step, wherein said amplifying stepcomprises contacting said sample with a pair of IS481 primers to producean IS481 amplification product if a B. pertussis IS481 nucleic acidmolecule is present in said sample, wherein said pair of IS481 primerscomprises a first IS481 primer and a second IS481 primer, wherein saidfirst IS481 primer consists of the sequence 5′-CCA GTT CCT CAA GGACGC-3′ (SEQ ID NO:1) and said second IS481 primer consists of thesequence 5′-GAG TTC TGG TAG GTG TGA GCG TA-3′ (SEQ ID NO:2), whereinsaid dye-binding step comprises contacting said IS481 amplificationproduct with a nucleic acid binding dye; and detecting the presence orabsence of binding of said nucleic acid binding dye to saidamplification product, wherein the presence of binding is indicative ofthe presence of B. pertussis in said sample, and wherein the absence ofbinding is indicative of the absence of B. pertussis in said sample. 21.The method of claim 20, wherein said nucleic acid binding dye isselected from the group consisting of SYBRGreenI®, SYBRGold®, andethidium bromide.
 22. The method of claim 21, further comprisingdetermining the melting temperature between said IS481 amplificationproduct and said nucleic acid binding dye, wherein said meltingtemperature confirms said presence or absence of said B. pertussis. 23.A method for detecting the presence or absence of B. parapertussis in abiological sample from an individual, said method comprising: performingat least one cycling step, wherein a cycling step comprises anamplifying step and a dye-binding step, wherein said amplifying stepcomprises contacting said sample with a pair of IS1001 primers toproduce an IS1001 amplification product if a B. parapertussis IS1001nucleic acid molecule is present in said sample, wherein said pair ofIS1001 primers comprises a first IS1001 primer and a second IS1001primer, wherein said first IS1001 primer consists of the sequence 5′-GGCGAT ATC AAC GGG TGA-3′ (SEQ ID NO:5) and said second IS481 primerconsists of the sequence 5′-CAG GGC AAA CTC GTC CAT C-3′ (SEQ ID NO:6),wherein said dye-binding step comprises contacting said IS1001amplification product with a nucleic acid binding dye; and detecting thepresence or absence of binding of said nucleic acid binding dye to saidamplification product, wherein the presence of binding is indicative ofthe presence of B. parapertussis in said sample, and wherein the absenceof binding is indicative of the absence of B. parapertussis in saidsample.
 24. The method of claim 23, wherein said nucleic acid bindingdye is selected from the group consisting of SYBRGreenI®, SYBRGold®, andethidium bromide.
 25. The method of claim 24, further comprisingdetermining the melting temperature between said IS1001 amplificationproduct and said nucleic acid binding dye, wherein said meltingtemperature confirms said presence or absence of said B. parapertussis.